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The invention relates to a method and apparatus for time-resolved measurements of lymphocyte function and cell aggregate structure using computer-automated microscopy.
Presently accepted methods for quantifying adhesion mechanics among cell aggregates are insensitive to the fine details of molecular pathways and structures which govern the cell aggregation process. Current methods of data presentation loose much information, and many physical characteristics of the aggregated cells which are important to the understanding of cell adhesion mechanics are never considered.
Known methods for quantifying cell aggregation often blur the distinction between aggregate shapes by use of rather simplistic, subjective scoring methods. For example, one known method for quantifying aggregate structure is through the use of a relative number scale presented below.
1+=&lt;10% of cells in small aggregates (5-10 cells/clump) PA1 2+=10-40% of cells aggregated (size varies) PA1 3+=40-90% of cells aggregated (size varies) PA1 4+=&gt;90% of cells aggregated, usually in medium to large clumps PA1 5+=approximately 100% of cells aggregated in large clusters
In order to eliminate the effects of personal bias, assays using this number scale are usually scored double-blind. While this has proved somewhat helpful in eliminating errors resulting from the subjectivity of the observer, the scoring method is still subject to human error and to variability between the perception of different observers. More importantly, this number scale does not reflect the potential differences in the physical dynamics or morphology that may exist between various adhesion events.
For example, using this number scoring method, adhesion induced in a human lymphoblastoid T-cell line, JURKAT, by anti-VLA-4 antibody was 5+, adhesion induced by the lectin PHA (phytohemagglutinin) was 5+, while adhesion induced by the phorbolester PMA (phorbol 12-myristate 13-acetate) was scored a 4+. These similar number scores occur despite the fact anti-VLA-4 induced aggregates are large round and compact, with very few free non-aggregated cells, aggregates induced by PHA are long-chained structures, and those induced by PMA are smaller, more loosely structured and contain far more non-aggregated cells. Stated otherwise, application of the known number scoring method to these three aggregate structures results in very similar numerical scores despite a markedly different appearance of the aggregates.
Another limitation of the simplistic numerical scoring method is the inability to establish rates of aggregate formation. Again, this results in a substantial loss of important information. For example, two antibodies that induce lymphocyte adhesion have recently been discovered; L25 (anti-VLA-4) and IC9 (ligand presently unknown). Both antibodies induce 5+ adhesion, but it has been observed that the rate of IC9-induced aggregation is considerably faster than that of L25-induced adhesion. A complete understanding of the different adhesion mechanisms induced by these two antibodies requires assessment of the relative rates of aggregation, an assessment which is not possible using presently available methodologies.